Process for lyophilized pharmaceutical formulation of a therapeutic protein

ABSTRACT

This invention concerns a process for making a lyophilized pharmaceutical formulation of a therapeutic protein, which comprises (a) providing a formulation of a bulk amount of the therapeutic protein, (b) measuring the concentration of the therapeutic protein in said bulk formulation, (c) adjusting the fill weight of the protein in said bulk formulation to achieve a fixed dose of the protein, and (d) lyophilizing the protein fill weight-adjusted formulation to achieve a final formulation in a container, wherein the product concentration post reconstitution with a fixed volume is within a predetermined acceptance range. The process is particularly suitable for formulations with low protein concentrations (e.g., 0.05 to 20 mg/mL).

FIELD OF THE INVENTION

This invention relates to biopharmaceuticals, particularly totherapeutic proteins, methods of use thereof, pharmaceuticalformulations thereof, and processes of making pharmaceuticalformulations. In particular, this invention relates to processes formaking lyophilized pharmaceutical formulations.

BACKGROUND OF THE INVENTION

In the past ten years, advances in technology have made it possible toproduce a variety of active molecules for pharmaceutical applications.As the nature of understanding of mechanisms of biological actionprogresses, these molecules can be designed for certain attributes,where small amounts of product can be efficacious.

Because these molecules can be larger and/or more complex thantraditional organic and inorganic drugs (i.e. possessing multiplefunctional groups in addition to complex three-dimensional structures),the formulation of such products poses special problems. For a productto remain biologically active, a formulation must preserve intact theconformational integrity of at least a core sequence of the protein'samino acids while at the same time protecting the protein's multiplefunctional groups from degradation. Degradation pathways for proteinscan involve chemical instability (i.e. any process which involvesmodification of the protein by bond formation or cleavage resulting in anew chemical entity) or physical instability (i.e. changes in the higherorder structure of the protein). Chemical instability can result fromdeamidation, racemization, hydrolysis, oxidation, beta elimination ordisulfide exchange. Physical instability can result from denaturation,aggregation, precipitation or adsorption, for example. The three mostcommon protein degradation pathways are protein aggregation, deamidationand oxidation, Cleland et al. (1993), Critical Reviews in TherapeuticDrug Carrier Systems 10(4): 307-377.

These designed molecules due to their synthetic nature are prevalentlylyophilized (freeze dried) as the presentation can provide unprovedshelf stability. Freeze-drying is a commonly employed technique forpreserving proteins which serves to remove water from the proteinpreparation of interest. Freeze-drying, or lyophilization, is a processby which the material to be dried is first frozen and then the ice orfrozen solvent is removed by sublimation in a vacuum environment. Anexcipient may be included in pre-lyophilized formulations to enhancestability during the freeze-drying process and/or to improve stabilityof the lyophilized product upon storage, Pikal, M. (1990), Biopharm.3(9)26-30 and Arakawa et al. (1991), Pharm. Res. 8(3):285-291.

A designed molecule with specific biological targets and thee resultingdosage requirements for product poses new problems for the manufacturingprocess. The current art involves a simple process where product isformulated to a targeted concentration and then filled into containersat a set volume.

SUMMARY OF THE INVENTION

This invention is directed to a process to generate lyophilized drugproduct. In particular, it relates to the formulation, fill, andassurance of the required amount of product present post-reconstitutionwith a fixed volume of diluent of a lyophilized product for use.

Provided in accordance with the present invention is a process formaking a lyophilized pharmaceutical formulation of a therapeuticprotein, which comprises:

-   -   a) providing a formulation of a bulk amount of the therapeutic        protein,    -   (b) measuring the concentration of the therapeutic protein in        said bulk formulation,    -   (c) adjusting the fill weight of the protein in said bulk        formulation to achieve a fixed dose of the protein, and    -   (d) lyophilizing the protein fill weight-adjusted formulation to        achieve a final formulation in a container,        wherein the product concentration post reconstitution with a        fixed volume is within a predetermined acceptance range

In the foregoing process, the protein concentration in the finalformulation is preferred to be less than or equal to about 20 or 2.5mg/mL, with about 0.5 mg/mL, about 0.05 mg/mL, about 18 mg/mL, about 20mg/mL, and about 21 mg/mL most preferred. Preferred therapeutic proteinsin the processes of this invention are romiplostim, blinatumomab,infliximab, trastuzumab, AMG 701, and AMG 330, AMG 701 and AMG 330 arebispecific single chain antibody constructs and other bispecific singlechain antibody constructs (e.g., bispecific T cell engagers) arepreferred therapeutic proteins in the processes of the invention.Preferred pharmaceutical excipients present in the formulation comprisesugars, with trehalose, sucrose and a hydrate of either most preferred.Preferred pharmaceutical excipients also comprise buffers, withhistidine, citric acid monohydrate, sodium phosphate, potassiumphosphate, and glutamic acid preferred. Preferred excipients furthercomprise surfactants, with polysorbate 20 and polysorbate 80 mostpreferred. Preferred excipients and therapeutic proteins used inaccordance with the processes of this invention appear in Table 1, withabout the preferred concentrations of each listed below each protein andexcipient.

TABLE 1 Preferred Formulation Components Bulking agent/ SolubilizingProtein Sugar Buffer agent Surfactant pH romi- Sucrose HistidineMannitol Polysorbate 5.0 plostim  2% w/v  10 mM  4% w/v 20  0.5 mg/mL0.004% w/v blina- Trehalose Citric acid — Polysorbate 7.0 tumomab 15%w/v monohydrate 80 55 mcg/mL  25 mM;   0.1% w/v L-lysine hydrochloride200 mM infliximab Sucrose Sodium — Polysorbate 7.2 20 ± 1.5 10% w/vphosphate 80 mg/mL  10 mM  0.01% w/v trastu- {dot over (a)},á- Histidine— Polysorbate 6.1 zumab trehalose 0.303 mg/mL 20  21 mg/mL dehydrateL-histidine 0.0840 19.1 mg/mL hydrochloride mg/mL monohydrate 0.470mg/mL AMG 701 Sucrose L-Glutamic — Polysorbate 4.2   1 mg/mL 9% w/v acid80 10 mM  0.010% w/v AMG 330 Sucrose Potassium SBE-CD Polysorbate 6.10.5 mg/mL  8% w/v phosphate 1% w/v 80 10 mM  0.010% w/v

Further in accordance with the present invention, the formulation maycomprise other excipients as described hereinbelow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a response surface map showing an example design space for alow dose product in which formulation and fill weight would follow atypical control strategy. The gray space represents whereinmethod/reconstitution variability would have a greater than 50%probability of having a failed protein concentration result. The largesquare shows the current operating range for formulation development.The smaller rectangle shows an effective operating range.

FIG. 2 is a response surface map showing an example design space for alow dose product in which osmolality is considered in addition to bulkconcentration and fill volume. The light gray region shows thestepped-in drug product concentration specification whereinmethod/reconstitution variability would have a greater than 50%probability of having a failed protein concentration result. The darkgray area shows the osmolality specification. The rectangle shows thedesign space range for the fill weight (x-axis) and the formulated bulkconcentration (y-axis) in-process control (IPC)/alert limit operationalrange (ALOR).

FIG. 3 is a response surface map showing an example of a viable but notfault-tolerant fill weight and formulation control strategy. The curveand two-headed arrow show the likely fill target error. The light grayarea represents the stepped-in drug product concentration specification,with the light gray area showing where method/reconstitution variabilitywould have a greater than 50% probability of having a failed proteinconcentration result. The dark gray area defines the osmolalityspecification. The rectangle shows the IPC/ALOR.

FIG. 4 is a response surface map showing an example of a fill weight andformulation control strategy with difficulty at the edge of rangecontrol, with an expected lower formulated bulk and lower fill weight.The left curve and two-headed arrow shows the likely fill target errorat a low fill volume. The right curve and two-headed arrow show thelikely fill target error at a higher fill volume. The light gray arearepresents the stepped-in drug product concentration specification, withthe light gray area showing where method/reconstitution variabilitywould have a greater than 50% probability of having a failed proteinconcentration result. The dark gray area defines the osmolalityspecification. The rectangle shows the IPC/ALOR.

FIG. 5 is a response surface map showing an example of a combinedstrategy of using the formulating bulk result to then adjust the fillweight set point based upon a total product dose in the vial target,resulting in a viable, fault-tolerant lyophilized drug process. Thecurve and two-headed arrow show the likely fill target error. The lightgray area defines the stepped-in drug product concentrationspecification as in FIGS. 1 to 4. The dark gray area defines theosmolality specification. The rhomboid shows the IPC/ALOR, cut off bythe osmolality specification at the highest fill volumes within theIPC/ALOR.

FIG. 6 shows normalized fill weights vs. product protein concentrationpost reconstitution as determined in accordance with Example 1hereinafter.

FIG. 7 shows normalized fill weights vs. osmolality as determined inaccordance with Example 1 hereinafter.

DETAILED DESCRIPTION OF THE INVENTION Definition of Terms

In the description that follows, a number of terms are used extensively.The following definitions are provided to facilitate understanding ofthe invention.

Unless otherwise specified, “a”, “an”, “the”, and “at least one” areused interchangeably and mean one or more than one. In addition, unlessotherwise required by context, singular terms shall include pluralitiesand plural terms shall include the singular.

As used herein, a “pharmaceutical formulation” or a “formulation” is asterile composition of (i) a pharmaceutically active drug, such as abiologically active protein, that is suitable for parenteraladministration (including but not limited to intravenous, intramuscular,subcutaneous, aerosolized, intrapulmonary, intranasal and intrathecaladministration) to a patient in need thereof and (ii) one or morepharmaceutically acceptable excipients, diluents, and other additivesdeemed safe by the Federal Drug Administration or other foreign nationalauthorities. Pharmaceutical formulations include liquid (e.g., aqueous)solutions that may be directly administered, and lyophilized powdersthat may be reconstituted into solutions by adding a diluent beforeadministration. The term “pharmaceutical formulation” specificallyexcludes, however, compositions for topical administration to patients,compositions for oral ingestion, and compositions for parenteralfeeding.

“Shelf life”, as used herein, means that the storage period during whichan active ingredient (e.g., an antibody) in a pharmaceutical formulationhas minimal degradation (e.g., not more than about 5% to 10%degradation) when the pharmaceutical formulation is stored underspecified storage conditions (e.g., 2-8° C.). Techniques for assessingdegradation vary depending on the identity of the protein in thepharmaceutical formulation. Exemplary techniques include size-exclusionchromatography (SEC)-HPLC to detect, for example, aggregation; reversephase (RP)-HPLC to detect, for example, protein fragmentation; ionexchange-HPLC to detect, for example, changes in the charge of theprotein; and mass spectrometry, fluorescence spectroscopy, circulardichroism (CD) spectroscopy, Fourier transform infrared spectroscopy(FT-IR), and Raman spectroscopy to detect protein conformationalchanges. All of these techniques can be used singly or in combination toassess the degradation of the protein in the pharmaceutical formulationand determine the shelf life of that formulation. The pharmaceuticalformulations of the present invention preferably exhibit not more thanabout 5 to 10% increases in degradation (e.g., fragmentation,aggregation or unfolding) over two years when stored at 2-6° C.

As used herein, “stable” formulations of biologically active proteinsare formulations that exhibit either (i) reduced aggregation and/orreduced loss of biological activity of at least 20% upon storage at 2-8°C. for at least 2 years compared with a control formula sample, or (ii)reduced aggregation and/or reduced loss of biological activity underconditions of thermal stress (e.g. 25° C. for 1 week to 12 weeks; 40° C.for 1 to 12 weeks; 52° C. for 7-8 days, etc.). In an embodiment, aformulation is considered stable when the protein in the formulationretains its physical stability, chemical stability and/or biologicalactivity.

A protein may be said to “retain its physical stability” in aformulation if, for example, it shows no signs of aggregation,precipitation and/or denaturation upon visual examination of colorand/or clarity, or as measured by UV light scattering or by sizeexclusion chromatography (SEC) or electrophoresis, such as withreference to turbidity or aggregate formation.

A protein may he said to “retain its chemical stability” in aformulation if, for example, the chemical stability at a given time issuch that no new chemical entity results from modification of theprotein by bond formation or cleavage. In a further embodiment, chemicalstability can be assessed by detecting and quantifying chemicallyaltered forms of the protein. Chemical alteration may involve, forexample, size modification (e.g., clipping), which can be evaluatedusing size exclusion chromatography, SDS-PAGE and/or matrix-assisted,laser desorption ionization/time-of-flight mass spectrometry (MALDI/TOFMS). Other types of chemical alteration include, for example, chargealteration (e.g., resulting from deamidation), which can be evaluated byion-exchange chromatography, Oxidation is another commonly seen chemicalmodification.

A protein may be said to “retain its biological activity” in apharmaceutical formulation relative to unmodified protein if, forexample, the percentage of biological activity of the formulated protein(e.g., an antibody) as determined by an assay (e,g., an antigen bindingassay) compared to the control solution is between either about 50% andabout 200%, about 60% and about 170%, about 70% and about 150%, about80% and about 125%, or about 90% and about 110%. In a furtherembodiment, a protein may be said to “retain its biological activity” ina pharmaceutical formulation, if, for example, without limitation, thebiological activity of the protein at a given time is at least 1%, 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95% or 100%.

As used herein, the terms “comprising” and “comprises” are intended tomean that the formulations and methods include the listed elements butdo not exclude other unlisted elements. The terms “consistingessentially of” and “consists essentially of,” when used to defineformulations and methods include the listed elements, exclude unlistedelements that alter the basic nature of the formulation and/or method,but do not exclude other unlisted elements. So a formulation consistingessentially of elements defined herein would not exclude trace amountsof other elements, such as contaminants from any isolation andpurification methods or pharmaceutically acceptable carriers (e.g.,phosphate buffered saline), preservatives, and the like, but wouldexclude, for example, additional unspecified amino acids. The terms“consisting of” and “consists of” when used to define formulations andmethods exclude more than trace elements of other ingredients andsubstantial method steps for administering the compositions describedherein. Embodiments defined by each of these transition terms are withinthe scope of this disclosure and the inventions embodied herein.

The term “isolated” as used herein refers to a protein (e.g., anantibody) that has been identified and separated and/or recovered from acomponent of its natural environment. Contaminant components of itsnatural environment are materials which would interfere with diagnosticor therapeutic uses for the protein, and may include enzymes, hormones,and other proteinaceous eir nonproteinaceous solutes. In preferredembodiments, the protein will be purified (1) to greater than 95% byweight of antibody as determined by the Lowry method, and mostpreferably more than 99% by weight, (2) to a degree sufficient to obtainat least 15 residues of N-terminal or internal amino acid sequence byuse of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGEwider reducing or nonreducing conditions using Coomassie blue or,preferahly silver stain. Isolated protein includes the protein in situwithin recombinant cells since at least one component of the protein'snatural environment will not be present. Ordinarily, however, isolatedprotein will be prepared by at least one purification step.

The invention concerns processes for pharmaceutical formulations oftherapeutic proteins such as antibodies. “Antibodies” (Abs) and thesynonym “immunoglobulins” (Igs) are glycoproteins having the samestructural characteristics. While antibodies exhibit binding specificityto a specific antigen, immunoglobulins include both antibodies and otherantibody-like molecules that lack antigen specificity. Polypeptides ofthe latter kind are, for example, produced at low levels by the lymphsystem and at increased levels by myelomas. Thus, as used herein, theterm “antibody” or “antibody peptide(s)” refers to an intact antibody,an antibody derivative, an antibody analog, a genetically alteredantibody, an antibody having a detectable label, an antibody thatcompetes for specific binding with an antibody disclosed in thisspecification, m an antigen-binding fragment (e.g., Fab, Fab′, F(ab′)₂,Fv, single domain antibody) thereof that competes with the intactantibody for specific binding and includes chimeric, humanized, fullyhuman, and bispecific antibodies. In certain embodiments,antigen-binding fragments are produced, for example, by recombinant DNAtechniques. In additional embodiments, antigen-binding fragments areproduced by enzymatic or chemical cleavage of intact antibodies.Antigen-binding fragments include, but are not limited to, Fab, Fab′,F(ab)², F(ab′)², Fv, and single-chain antibodies.

The term “intact antibodies” as used herein refers to antibodiescomprising two heavy chains and two light chains. The term thus includeswithout limitation fully human antibodies, genetically alteredantibodies, bispecific antibodies, and antibody derivatives providedsuch antibodies comprised two heavy chains and two light chains.

The term “monoclonal antibody” as used herein is not limited toantibodies produced through hybridoma technology. The term “monoclonalantibody” refers to an antibody that is derived from a single clone,including any eukaryotic, prokaryotic, or phage clone, and not themethod by which it is produced.

The monoclonal antibodies and antibody constructs formulated inaccordance with the present invention specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is/are identical with or homologous to corresponding sequencesin antibodies derived from another species or belonging to anotherantibody class or subclass, as well as fragments of such antibodies, solong as they exhibit the desired biological activity (U.S. Pat. No.4,816,567; Morrison et al. (1984). Proc. Natl. Acad. Sci. USA, 81:6851-6855). Chimeric antibodies of interest herein include “primitized”antibodies comprising variable domain antigen-binding sequences derivedfrom a non-human primate (e.g., Old World Monkey, Ape, etc.) and humanconstant region sequences. A variety of approaches for making chimericantibodies have been described. See e.g., Morrison et al. (1985), Proc.Natl. Acad. Sci. U.S.A., 81:6851; Talceda et al. (1985), Nature 314:452,Cabilly et al., U.S. Pat. No. 4,816,567; Boss et al., U.S. Pat. No.4,816,397; Taniguchi et al., EP 0171496; EP 0173494; and GB 2177096.

The monoclonal antibodies and antibody constructs formulated inaccordance with the present invention specifically include antibodiesreferred to as “human” or “fully human.” The terms “human antibody” and“fully human antibody” each refer to an antibody that has an amino acidsequence of a human immunoglobulin, including antibodies isolated fromhuman immunoglobulin, libraries or from animals transgenic for one ormore human immunoglobulins and that do not express endogenousimmunoglobulins; for example, Xenomouse® antibodies and antibodies asdescribed by Kucherlapati et al, in U.S. Pat. No. 5,939,598.

The term “genetically altered antibodies” means antibodies wherein theamino acid sequence has been varied from that of a native antibody.Because of the relevance of recombinant DNA techniques in the generationof antibodies, one need not be confined to the sequences of amino acidsfound in natural antibodies; antibodies can be redesigned to obtaindesired characteristics. The possible variations are many and range fromchanges to just one or a few amino acids to complete redesign of, forexample, the variable and/or constant region. Changes in the constantregion will, in general, be made in order to improve or altercharacteristics, such as complement fixation, interaction with membranesand other effector functions, as well as manufacturability andviscosity. Changes in the variable region will be made in order toimprove the antigen binding characteristics.

A “Fab fragment” is comprised of one light chain and the C_(H1) andvariable regions of one heavy chain. The heavy chain of a Fab moleculecannot form a disulfide bond with another heavy chain molecule.

A “Fab fragment” contains one light chain and one heavy chain thatcontains more of the constant region, between the C_(H1) and C_(H2)domains, such that an interchain disulfide bond can be formed betweentwo heavy chains to form a F(ab′)₂ molecule.

A “F(ab′)₂ fragment” contains two light chains and two heavy chainscontaining a portion of the constant region between the C_(H1) andC_(H2) domains, such that an interchain disulfide bond is formed betweentwo heavy chains.

The terms “Fv fragment” and “single chain antibody” refer topolypeptides containing antibody variable regions from both heavy andlight chains but lacking constant regions. Like a whole antibody, it isable to bind selectively to a specific antigen. With a molecular weightof only about 25 kDa, Fv fragments are much smaller than commonantibodies (150-160 kD) which are composed of two heavy protein chainsand two light chains, and even smaller than Fab fragments (about 50 kDa,one light chain and half a heavy chain).

A “single domain antibody” is an antibody fragment consisting of asingle domain Fv unit, e.g., V_(H) or V_(L). Like a whole antibody, itis able to bind selectively to a specific antigen. With a molecularweight of only 12-15 kDa, single-domain antibodies are much smaller thancommon antibodies (150-160 kDa) which are composed of two heavy proteinchains and two light chains, and even smaller than Fab fragments (about50 kDa, one light chain and half a heavy chain) and single-chainvariable fragments (about 25 kDa, two variable domains, one from a lightand one from a heavy chain). The first single-domain antibodies wereengineered from heavy-chain antibodies found in camelids. Although mostresearch into single-domain antibodies is currently based on heavy chainvariable domains, light chain variable domains and nanobodies derivedfrom light chains have also been shown to bind specifically to targetepitopes.

The term “bispecific” as used herein refers to an antibody constructwhich is “at least bispecific”, i.e., it comprises at least a firstbinding domain and a second binding domain, wherein the first bindingdomain binds to one antigen or target (e.g., CD3), and the secondbinding domain binds to another antigen or target (e.g., BCMA; e.g., CD33). Accordingly, antibody constructs according to the inventioncomprise specificities for at least two different antigens or targets.The term “bispecific antibody construct” of the invention alsoencompasses multispecific antibody constructs such as trispecificantibody constructs, the latter ones including three binding domains, orconstructs having more than three (e.g. four, five . . . )specificities.

Given that the antibody constructs according to the invention are (atleast) bispecific, they do not occur naturally and they are markedlydifferent from naturally occurring products. A “bispecific” antibodyconstruct or immunoglobulin is hence an artificial hybrid antibody orimmunoglobulin having at least two distinct binding sites with differentspecificities. Bispecific antibody constructs can be produced by avariety of methods including fusion of hybridomas or linking of Fab′fragments. See, e.g., Songsivilai & Lachmann, Clin. Exp. Immunol.79:315-321 (1990).

The at least two binding domains and the variable domains of theantibody construct of the present invention may or may not comprisepeptide linkers (spacer peptides). The term “peptide linker” comprisesin accordance with the present invention an amino acid sequence by whichthe amino acid sequences of one (variable and/or binding) domain andanother (variable and/or binding) domain of the antibody construct ofthe invention are linked with each other. An essential technical featureof such peptide linker is that it does not comprise any polymerizationactivity. Among the suitable peptide linkers are those described U.S.Pat. Nos. 4,751,180 and 4,935,233 or WO 88/09344. The peptide linkerscan also be used to attach other domains or modules or regions (such ashalf-life extending domains) to the antibody construct of the invention.

In the event that a linker is used, this linker is preferably of alength and sequence sufficient to ensure that each of the first andsecond domains can, independently from one another, retain theirdifferential binding specificities. For peptide linkers which connectthe at least two binding domains (or two variable domains) in theantibody construct of the invention, those peptide linkers are preferredwhich comprise only a few number of amino acid residues, e.g. 12 aminoacid residues or less. Thus, peptide linkers of 12, 11, 10, 9, 8, 7, 6or 5 amino acid residues are preferred. An envisaged peptide linker withless than 5 amino acids comprises 4, 3, 2 or one amino acid(s), whereinGly-rich linkers are preferred. A particularly preferred “single” aminoacid n the context of said “peptide linker” is Gly. Accordingly, saidpeptide linker may consist of the single amino acid Gly. Anotherpreferred embodiment of a peptide linker is characterized by the aminoacid sequence Gly-Gly-Gly-Gly-Ser, i.e. Gly₄Ser (SEQ ID NO: 1), orpolymers thereof, i.e. (Gly₄Ser)x, where x is an integer of 1 or greater(e.g. 2 or 3). Preferred linkers are depicted in SEQ ID NOs: 1-9. Thecharacteristics of said peptide linker, which comprise the absence ofthe promotion of secondary structures, are known in the art and aredescribed e.g. in Dall'Aequa et al. (Biochem. (1998) 37, 9266-9273),Cheadle et al. (Mol. Immunol. (1992) 29, 21-30) and Raag and Whitlow(FASB (1995) 9(1), 73-80). Peptide linkers which furthermore do notpromote any secondary structures are preferred. The linkage of saiddomains to each other can be provided, e.g., by genetic engineering, asdescribed in the examples. Methods for preparing fused and operativelylinked bispecific single chain constructs and expressing them inmammalian cells or bacteria are well-known in the art (e.g., WO 99/54440or Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory. Press, Cold Spring Harbor, New York, 2001).

According to a particularly preferred embodiment and as documented inthe appended examples, the AMG 701 and AMG 330 antibody constructs ofthe invention are each a “bispecific single chain antibody construct”,more preferably a bispecific “single chain Fv” (scFv). Although the twodomains of the Fv fragment, VL and VH, are coded for by separate genes,they can be joined, using recombinant methods, by a synthetic linker—asdescribed hereinbefore—that enables them to be made as a single proteinchain in which the VL and VH regions pair to form a monovalent molecule;see e.g., Huston et al. (1988) Proc. Natl. Acad. Sci USA 85:5879-5883).These antibody fragments are obtained using conventional techniquesknown to those with skill in the art, and the fragments are evaluatedfor function in the same manner as are whole or full-length antibodies.A single-chain variable fragment (scFv) is hence a fusion protein of thevariable region of the heavy chain (VH) and of the light chain (VL)immunoglobulins, usually connected with a short linker peptide of aboutten to about 25 amino acids, preferably about 15 to 20 amino acids. Thelinker is usually rich in glycine for flexibility, as well as serine orthreonine for solubility, and can either connect the N-terminus of theVH with the C-terminus of the VL, or vice versa. This protein retainsthe specificity of the original immunoglobulin, despite removal of theconstant regions and introduction of the linker.

Bispecific single chain molecules are known in the art and are describedin WO 99/54440, Mack, J. Immunol. (1997), 158, 3965-3970, Mack, PNAS,(1995), 92, 7021-7025, Kufer, Cancer Immunol. Immunother., (1997), 45,193-197, Löffler, Blood, (2000), 95, 6, 2098-2103, Brühl. Immunol.,(2001), 166, 2420-2426, Kipriyanov, J. Mol, Biol., (1999). 293, 41-56,Techniques described for the production of single chain antibodies (see,inter alia, U.S. Pat. No. 4,946,778) can be adapted to produce singlechain antibody constructs specifically recognizing (an) electedtarget(s).

Bivalent (also called divalent) or bispecific single-chain variablefragments (bi-scFvs or di-scFvs having the format (scFv)₂ can beengineered by linking two scFv molecules (e.g. with linkers as describedhereinbefore). If these two scFv molecules have the same bindingspecificity, the resulting molecule will preferably be called bivalent(i.e. it has two valences for the same target epitope). If the two scFvmolecules have different binding specificities, the resulting (scFv)₂molecule will preferably be called bispecific. The linking can be doneby producing a single peptide chain with two VH regions and two VLregions, yielding tandem scFvs (see e.g. Kufer P. et al., (2004) Trendsin Biotechnology 22(5):238-244). Another possibility is the creation ofscFv molecules with linker peptides that are too short for the twovariable regions to fold together (e.g. about five amino acids), forcingthe scFvs to dimerize. This type is known as diabodies (see e.g.Hollinger, Philipp et al., (July 1993) Proceedings of the NationalAcademy of Sciences of the United States of America 90 (14): 6444-8.).

As described herein above, the invention provides a preferred embodimentwherein the antibody construct is in a format selected from the groupconsisting of (scFv)₂, scFv-single domain mAb, diabodies and oligomersof any of the those formats.

According to an also preferred embodiment of the antibody construct ofthe invention the heavy chain (VH) and of the light chain (VL) of abinding domain binding either to the target antigen CD3 and CD33 or BCMAare not directly connected via an above described peptide linker but thebinding domain is formed due to the formation of a bispecific moleculeas described for the diabody. Thus, the VH Chain of the CD3 bindingdomain may be used to the VL the CD33 or BCMA binding domain via suchpeptide linker, while the VH chain of the CD3 binding domain is fused tothe VL of the CD33 o BCMA binding domain via such peptide linker.

The terms “amino-terminal” and “carboxyl-terminal” and their shortenedforms “N-terminus” and “C-terminus” are used herein to denote positionswithin polypeptides. Where the context allows, these terms are used withreference to a particular sequence or portion of a polypeptide to denoteproximity or relative position. For example, a certain sequencepositioned carboxyl-terminal to a reference sequence within apolypeptide is located proximal to the carboxyl terminus of thereference sequence, but is not necessarily at the carboxyl terminus ofthe complete polypeptide.

As used herein, the term “amino acid” refers to either natural and/orunnatural or synthetic amino acids, including glycine and both the D andL optical isomers, amino acid analogs and peptidoinimetics, includingwithout limitation N-acetyl analogs of D or L optical isomers (e.g.,N-acetyl arginine). In some aspects, the term amino acid refers tomonomeric amino acids.

Generally, nomenclatures used in connection with, and techniques of,cell and tissue culture, molecular biology, immunology, microbiology,genetics and protein and nucleic acid chemistry and hybridizationdescribed herein are those well known and commonly used in the art. Themethods and techniques of the present invention are generally performedaccording to conventional methods well known in the art and as describedin various general and more specific references that are cited anddiscussed throughout the present specification unless otherwiseindicated. See, for example, Sambrook et al. (2001), Molecular Cloning:A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. and Ausubel et al. (1992), Current Protocols inMolecular Biology, Greene Publishing Associates, and Harlow and Lane(1990), Antibodies: A Laboratory Manual, Cold Spring Harbor LaboratoryPress. Cold Spring Harbor, N.Y. Any enzymatic reactions and purificationtechniques are performed according to manufacturer's specifications, ascommonly accomplished in the art or as described herein. The terminologyused in connection with, and the laboratory procedures and techniquesof, analytical chemistry, synthetic organic chemistry, and medicinal andpharmaceutical chemistry described herein are those well known andcommonly used in the art. Standard techniques can be used for chemicalsyntheses, chemical analyses, pharmaceutical preparation, formulation,and delivery, and treatment of patients.

Use of Intermediate Formulated Concentrations

The goal of protein formulation is to transform a highly purified,recombinant protein solution (drug substance) into a stable, efficaciousbiopharmaceutical dosage form. Kamerzell et al, (2011), 63(13): 1118-59(incorporated by reference). The first step, often called preformulationcharacterization, involves determining the protein's physicochemicalproperties and pathways of instability, which allows for design offormulations containing various excipients to ensure protein stabilityunder defined storage conditions. At the same time, analytical methodsto monitor the protein's physicochemical integrity and biologicalactivity under formulation conditions (e.g., in the presence ofexcipients) need to be developed, along with specifications to defineacceptable limits to any changes in these parameters. Specificformulations at different protein concentrations with targeted levels ofvarious pharmaceutical excipients are then experimentally tested toensure stability, solubility and tonicity over the shelf life. Inaddition, the primary container is selected (e.g., vial, cartridge orprefilled syringe) to store the protein-excipient mixture and facilitateparenteral administration by the patient or a medical professional. Theentire biopharmaceutical drug or vaccine dosage form (protein,excipients, primary container, and delivery device) must be designed forboth scalability, to allow for commercial manufacturing under sterileconditions, and to meet all regulatory guidelines for the production andtesting of biopharmaceutical dosage forms for human use.

Formulation development generally starts with identifying the right pHand buffer system through a biophysical screening strategy. Buffers areadded to the protein solution to stabilize pH, which in turn stabilizesthe protein because a protein's stability is generally linked to acharacteristic, narrow pH range. See Garidel and Bassarab (2009),“Impact of formulation design on stability and quality,” in: Quality forBiologics: Critical Quality Attributes, Process and Change Control,Production Variation, Characterisation Impurities and Concerns, BiopharmKnowledge Publishing, London, UK, pp. 94-113 (incorporated byreference). Other formulation excipients, such as stabilizers (e.g.,sugars), bulking agents (e.g., mannitol), and surfactants (e.g.,polysorbate 20) are then added to the buffered protein solution. For alyophilized formulation, such a formulation mixture in liquid form isthen lyophilized.

In lyophilization as in other drug product processes, a key control istherapeutic protein fill weight to deliver the required dose to thepatient. For higher concentration products, a simple strategy ofcontrolling the product concentration at the formulation stage, followedby fill weight control as part of the fill process, is adequate. Forvery low dose delivery in micro or even milligrams of product, however,this simple strategy for ensuring dose delivery begins to have problemsin ensuring that the patient can receive the required dose. Relatedconcerns are that the product label may not reflect the product withinthe container and that one may have difficulty extracting the materialfor dosing. A need exists, therefore, for a process providing greatercontrol over the fill weight of a therapeutic protein in a lyophilizedpharmaceutical formulation.

As noted above, a simple strategy of controlling the productconcentration at the formulation stage, followed then by fill weightcontrol as part of the fill process is adequate for higherconcentrations of therapeutic protein but can lead to problems in lowerconcentrations. The design space for fill weight control for low doseproduct when therein weight and formulation target are controlledseparately shows a higher variability, with a greater likelihood theproduct will not meet the release specification. Due to manufacturingvariability, precisely controlling the formulated bulk is not achievabledue to the scale of the process and the inherent variability of theequipment in manufacturing. Another issue is that increased variabilityis inherent in the lyophilized product because the product must bereconstituted, which is also a variable process.

A typical control strategy (shown in FIG. 1) would have a greater than50% probability of having a failed protein concentration result. In FIG.1, the observed overall variability of lyophilized homogeneity data is0.01 mg/mL. To assure minimal capability, the average operating rangemust be constrained such that it corresponds to a specification range“stepped-in” by 3*0.01=0.03 mg/mL. A process that is targeted within thewhite space has quality of less than 0.1% of Out of Specification (OOS)vials per batch. A process that is targeted within the shaded area, incontrast, has greater OOS rates. As cart be seen from FIG. 1, thecurrent operating range (large square) contains edges of failure of thedesired formulation. An effective operating range (smaller rectangle inFIG. 1), however, requires tighter fill tolerances than can be achievedas part of both the formulation variability and the filler weightcontrol.

Thus, a process that also needs to consider other product qualityattributes (e.g., osmolality, pH) as well as protein concentration mightnot be producible or viable by the standard methodology. FIG. 2 shows anexample design space in which an osmolality specification is considered.The gray coloring shows the extensive failure conditions within theIPC/alert limit operation range.

Standard methodology can also result in a viable but not fault-tolerantfill weight and formulation control strategy (see FIG. 3). As in FIG. 1,the IPC/ALOR contains areas that do not meet the drug productconcentration specification. Within the area that does meet the drugproduct concentration specification (white area within the IPC/ALOR),there is very little room for error in the fill target. Thus, as shownin FIG. 3, standard methodology may result in a process with difficultyat the edge of range control, viable but without sufficient faulttolerance.

An increase in fill volume might not be enough of a process variation tocreate a viable formulation with adequate fault tolerance. FIG. 4 is aresponse surface map in which fill volume is increased. The fill targetrange may be brought within the IPC/ALOR and product specifications butfault tolerance may remain inadequate. The fill weight range over theallowable range of the formulated bulk concentration has insufficientfault tolerance.

If the formulated bulk result is used to then adjust the therapeuticprotein fill weight, the operating range may be changed enough to enablea formulation that is both viable and sufficiently fault-tolerant. Asshown in FIG. 5, the modified operating range (IPC/ALOR) allows for theexpected fill target range to be well within the concentration andosmolality specifications. In this way, the fill target range can beprovided within the concentration and other specifications withsufficient fault tolerance.

To date, this technique has been used for a low dose stock keeping unit(SKU) for romiplostim (Nplate®) low dose SKU, blinatumomab (Blincyto®),infliximab, and trastuzumab. Details on use of this technique appear inthe Working Examples hereinafter.

Excipients in General

One challenge in formulations is stabilizing the product against thestresses of manufacturing, shipping and storage, which can beaccomplished by certain formulation excipients. In general, excipientscan be classified on the basis of the mechanisms by which they stabilizeproteins against various chemical and physical stresses. Some excipientsalleviate the effects of a specific stress or regulate a particularsusceptibility of a specific protein. Other excipients more generallyaffect the physical and covalent stabilities of proteins.

Common excipients of liquid and lyophilized protein formulations appearin Table 2 (see Kamerzell et al. (2011), Advanced Drug Delivery Rev.63(13): 1118-59).

TABLE 2 Excipient components of protein formulations Excipient ComponentFunction Examples Buffers Maintaining pH of solution Citrate Buffer-ionspecific interactions Succinate with protein Acetate Glutamate AspartateHistidine Phosphate Tris Glycine Sugars and Stabilizing protein Sucrosecarbo- Tonicifying agents Trehalose hydrates Carrier for inhaled drugs(lactose) Sorbitol Dextrose solutions during IV Mannitol administrationGlucose Lactose Cyclodextrin derivatives Stabilizers Enhancing productelegance and Mannitol and bulking preventing blowout Glycine agentsProviding structural strength to a lyo cake Osmolytes Stabilizingagainst environmental Sucrose stress Trehalose (temperature,dehydration) Sorbitol Glycine Proline Glutamate Glycerol Urea Aminoacids Specific interactions with protein Histidine Antioxidant (His,Met) Arginine Buffering, tonicifying Glycine Proline Lysine MethionineAa mixtures (e.g., glu/arg) Proteins and Competitive inhibitors ofprotein HSA polymers adsorption PVA Bulking agents for lyophilizationPVP Drug delivery vehicles PLGA PEG Gelatin Dextran Hydroxyethyl starchHEC CMC Anti-oxidants Preventing oxidative protein damage Reducingagents Metal ion binders (if a metal is included Oxygen scavengers as aco-factor or is required for Free radical protease activity) scavengersFree radical scavengers Chelating agents (e.g., EDTA, EGTA, DTPA)Ethanol Metal ions Protein co-factors Magnesium Coordination complexes(suspensions) Zinc Specific Stabilizers of native conformation Metalsligands against stress-induced unfolding Ligands Providing conformationflexibility Amino acids Polyanions Surfactants Competitive inhibitor ofPolysorbate 20 protein adsorption Polysorbate 80 Competitive inhibitorof Poloxamer 188 protein surface denaturation Anionic surfactantsLiposomes as drug delivery vehicles (e.g., Inhibitor of aggregationduring sulfonates and lyophilization sulfosuccinates) Reducer ofreconstitution times of Cationic lyophilized products surfactantsZwitterionic surfactants Salts tonicifying agents NaCl stabilizing ordestabilizing agents for KCl proteins, especially anions NaSO₄Preservatives Protection against microbial growth in Benzyl alcoholformulation M-cresol Phenol

Other excipients are known in the art and can be found in Powell et al.(1998), “Compendium of Excipients for Parenteral Formulations,” PDA J.Pharm. Sci. Tech., 52:238-311, which is hereby incorporated byreference.

Given the teachings and guidance provided herein, those skilled in theart will know what amount or range of excipient can be included in anyparticular formulation to achieve a biopharmaceutical formulation of theinvention. For example, the amount and type of a salt to be included ina biopharmaceutical formulation of the invention can be selected basedon the desired osmolality (i.e., isotonic, hypotonic or hypertonic) ofthe final solution as well as the amounts and osmolality of othercomponents to he included in the formulation. Similarly, byexemplification with reference to the type of polyol or sugar includedin a formulation, the amount of such an excipient will depend on itsosmolality.

Those skilled in the art can determine what amount or range of excipientcan be included in any particular formulation to achieve abiopharmaceutical formulation of the invention that promotes retentionin stability of the biopharmaceutical. For example, the amount and typeof a salt to be included, in a biopharmaceutical formulation of theinvention can be selected based on to the desired osmolality (i.e.,isotonic, hypotonic or hypertonic) of the final solution as well as theamounts and osmolality of other components to be included in theformulation. Similarly, by exemplification with reference to the type ofpolyol or sugar included in a formulation, the amount of such anexcipient will depend on its osmolality.

About 5% (weight/volume) sorbitol, for example, can achieve isotonicitywhile about 9% (weight/volume) of a sucrose excipient is needed toachieve isotonicity. Selection of the amount or range of concentrationsof one or more excipients that can be included within abiopharmaceutical formulation of the invention has been exemplifiedabove by reference to salts, polyols and sugars. However, those skilledin the art will understand that the considerations described herein andfurther exemplified by reference to specific excipients are equallyapplicable to all types and combinations of excipients including, forexample, salts, amino acids, other tonicity agents, surfactants,stabilizers, bulking agents, cryoprotectants, lyoprotectants,anti-oxidants, metal ions, chelating agents and/or preservatives.

Further, where a particular excipient is reported in a formulation by,e.g., percent (%) w/v, those skilled in the art will recognize that theequivalent molar concentration of that excipient is also contemplated.

Those having ordinary skill in the art would recognize that theconcentrations of the aforementioned excipients share an interdependencywithin a particular formulation. By way of example, the concentration ofa bulking agent may be lowered where, for example, there is a highprotein/peptide concentration or a high stabilizing agent concentration.In addition, a person having ordinary skill in the art would recognizethat, in order to maintain the isotonicity of a particular formulationin which there is no bulking agent, the concentration of a stabilizingagent would be adjusted accordingly (i.e., a “tonicifying” amount ofstabilizer would be used).

Buffers

Solution pH affects the chemical integrity of a protein's amino acidresidues (e.g., As a deamidation and Met oxidation) and maintenance ofits higher order structure. Those skilled in the art thus use, bufferingagents to control solution pH and optimize protein stability. Maximalstability of a protein drug is usually within a narrow pH range. Severalapproaches (e.g., accelerated stability studies and calorimetricscreening studies) are useful for this purpose (Remmele et al. (1999),Biochemistry, 38(16): 5241-7). Once a formulation is finalized, the drugproduct must be manufactured and maintained within a predefinedspecification throughout its shelf-life. Hence, buffering agents arealmost always employed to control pH in the formulation.

Organic acids, phosphates and Tris have been employed routinely asbuffers in protein formulations (see Table 3). The buffer capacity ofthe buffering species is maximal at a pH equal to the pKa and decreasesas pH increases or decreases away from this value. Ninety percent of thebuffering capacity exists within one pH unit of its pKa. Buffer capacityalso increases proportionally with increasing buffer concentration.

TABLE 3 Commonly used buffering agents and their pK_(a) values BufferpKa Example Drug Product Acetate 4.8 Neupogen ®, Neulasta ® SuccinatepK_(a1) = 4.8, pK_(a2) = 5.5 Actimmune ® Citrate pK_(a1) = 3.1, pK_(a2)= 4.8, Humira ® pK_(a3) = 6.4 Histidine 6.0 Xolair ® (imidazole)phosphate pK_(a1) = 2.15, pK_(a2) = 7.2, Enbrel ® (liquid formulation)pK_(a3) = 12.3 Tris 8.1 Leukine ®

In addition to the foregoing, some therapeutic proteins may beself-buffering at a pharmaceutically relevant concentration.Formulations of such proteins might not need to include a conventionalbuffer at all. See US patent application 2012/0028877, which is herebyincorporated by reference.

Sugars and Carbohydrates

Sugars are frequently used to stabilize proteins in both liquid andlyophilized formulations. Disaccharides such as sucrose and trehaloseare thought to stabilize proteins by preferential hydration at highconcentrations in the liquid state and by specific interactions withproteins and formation of viscous glassy matrices in the solid state.Sugar molecules can increase the viscosity of monoclonal antibodysolutions, presumably due to a preferential hydration mechanism. Sugaralcohols such as sorbitol can stabilize proteins in solution and in thelyophilized state. Mannitol is often used as a bulking agent inlyophilized formulations. Lactose is used as a carrier molecule forinhaled formulations of proteins. Cyclodextrin derivatives can stabilizeproteins in liquid formulations of antibodies, vaccine antigens, andsuch smaller proteins as growth factors, interleukin-2 and insulin.Stabilizers and bulking agents.

Bulking agents are typically used in lyophilized formulations to enhanceproduct elegance and to prevent blowout. Conditions in the formulationare generally designed so that the bulking agent crystallizes out of thefrozen amorphous phase (either during freezing or annealing above theTg′) giving the cake structure and bulk. Mannitol and glycine areexamples of commonly used bulking agents.

Stabilizers include a class of compounds that can serve ascryoprotectants, lyoprotectants, and glass forming agents.Cryoprotectants act to stabilize proteins during freezing or in thefrozen state at low temperatures (P. Cameron, ed., Good PharmaceuticalFreeze-Drying Practice, Interpharm Press, Inc., Buffalo Grove, Ill.,(1997)). Lyoprotectants stabilize proteins in the freeze-dried soliddosage form by preserving the native like conformational properties ofthe protein during dehydration stages of freeze-drying. Glassy stateproperties have been classified as “strong” or “fragile” depending ontheir relaxation properties as a function of temperature. It isimportant that cryoprotectants, lyoprotectants, and glass forming agentsremain in the same phase with the protein in order to impart stability.Sugars, polymers, and polyols fall into this category and can sometimesserve all three roles.

Polyols encompass a class of excipients that includes sugars, (e.g.mannitol, sucrose, sorbitol), and other polyhydric alcohols (e.g.,glycerol and propylene glycol). The polymer polyethylene glycol (PEG) isincluded in this category. Polyols are commonly used as stabilizingexcipients and/or isotonicity agents in both liquid and lyophilizedparenteral protein formulations. With respect to the Hofmeister series,the polyols are kosmotropic and are preferentially excluded from theprotein surface. Polyols can protect proteins from both physical andchemical degradation pathways. Preferentially excluded co-solventsincrease the effective surface tension of solvent at the proteininterface whereby the most energetically favorable protein conformationsare those with the smallest surface areas.

Mannitol is a popular bulking agent in lyophilized formulations becauseit crystallizes out of the amorphous protein phase during freeze-dryinglending structural stability to the cake (e.g. Leukine®, Enbrel®—Lyo,Betaseron®). It is generally used in combination with a cryo and/orlyoprotectant like sucrose. Because of the propensity of mannitol tocrystallize under frozen conditions, sorbitol and sucrose are thepreferred tonicity agents/stabilizers in liquid formulations to protectthe product against freeze-thaw stresses encountered during transport orwhen freezing bulk prior to manufacturing. Sorbitol and sucrose are farmore resistant to crystallization and therefore less likely to phaseseparate from the protein. It is interesting to note that while mannitolhas been used in tonicifying amounts in several marketed liquidformulations such as Actimmune®, Fortco®, and Rebif®, the product labelsof these drugs carry a ‘Do Not Freeze’ warning. The use of reducingsugars (containing free aldehyde or ketone groups) such as glucose andlactose should be avoided because they can react and glycate surfacelysine and arginine residues of proteins via the Maillard reaction ofaldehydes and primary amines (Chevalier F. et al., Nahrung, 46(2): 58-63(2002); Humeny A, et al., J. Agric Food Chem. 50(7): 2153-60 (2002)).Sucrose can hydrolyze to fructose and glucose under acidic conditions(Kautz C. F. and Robinson A. L., JACS, 50(4) 1022-30 (1928)), andconsequently may cause glycation,

In particular embodiments of the present compositions, a stabilizer (ora combination of stabilizers) is added to a lyophilization formulationto prevent or reduce lyophilization-induced or storage-inducedaggregation and chemical degradation. A hazy or turbid solution uponreconstitution indicates that the protein has precipitated. The term“stabilizer” means an excipient capable of preventing aggregation orother physical degradation, as well as chemical degradation (forexample, autolysis, deamidation, oxidation, etc.) in an aqueous andsolid state. Stabilizers that are conventionally employed inpharmaceutical compositions include, but are not limited to, sucrose,trehalose, mannose, maltose, lactose, glucose, raffinose, cellobiose,gentiobiose, isomaltose, arabinose, glucosamine, fructose, mannitol,sorbitol, glycine, arginine HCL, poly-hydroxy compounds, includingpolysaccharides such as dextran, starch, hydroxyethyl starch,cyclodextrins, N-methyl pyrollidene, cellulose and hyaluronic acid,sodium chloride. Carpenter et al. (1991), Develop. Biol. Standard74:225.

Osmolytes

Osmolytes currently used as protein formulation excipients are listed inTable 2. Other osmolytes commonly found in nature that may be useful asexcipients include taurine, betaine, trimethylamine N-oxide (TMAO),choline-O-sulfate, and sarcosine.

Proteins and Polymers

Protein-based excipients add complexity to the formulation, especiallyin developing analytical methods to monitor the stability of theprotein-based drug or vaccine in the presence of a protein-basedexcipient. Polymers have been evaluated as excipients (e.g., as bulkingagents) in lyophilized protein formulations. Controlled releaseformulations of protein drugs and vaccines are being studied in whichproteins are formulated with polymers such as poly(lactic-co-glycolicacid) (PLGA) and polyethyletie glycol (PEG). Many additionalwater-soluble polymers (e.g., hydroxyethyl cellulose (HEC),carboxymethyl cellulose (CMC)) have been utilized for topicalformulations of protein drugs.

PEG can stabilize proteins by two different temperature-dependentmechanisms. At lower temperatures, it is preferentially excluded fromthe protein surface but has been shown to interact with the unfoldedform of the protein at higher temperature given its amphipathic nature(Lee and Lee (1987), Biochemistry, 26(24): 7813-9). It may protectproteins via preferential exclusion at lower temperatures but possiblyby reducing the number of productive collisions between unfoldedmolecules at higher temperatures. PEG is also a cryoprotectant and hasbeen employed in Recombinate®, a lyophilized formulation of recombinantAntihemophilic Factor.

Anti-Oxidants

Many different sources may oxidize protein residues. Oxidative proteindamage can be minimized by carefully controlling the manufacturingprocess and storage of the product, including such factors asatmospheric oxygen, temperature, light exposure, and chemicalcontamination. Where such controls are inadequate, anti-oxidantexcipients can be included in the formulation.

The most commonly used pharmaceutical antioxidant excipients arereducing agents, oxygen/free-radical scavengers, or chelating agents.Antioxidants in therapeutic protein formulations must be water-solubleand remain active throughout the product shelf-life. Reducing agents andoxygen/free-radical scavengers work by ablating active oxygen species insolution. Chelating agents (e.g., EDTA) can be effective by bindingtrace metal contaminants that promote free-radical formation. In theliquid formulation of acidic fibroblast growth factor, for example, EDTAinhibits metal ion-catalyzed oxidation of cysteine residues. EDTA hasbeen used in marketed products like Kineret® and Ontak®.

Metal Ions

In general, transition metal ions are undesired in protein formulationsbecause they can catalyze physical and chemical degradation reactions inproteins. Specific metal ions are included in formulations, however,when they act as co-factors to proteins. Metal ions may also be used insuspension formulations of proteins where they form coordinationcomplexes (e.g., zinc suspension of insulin). The use of magnesium ions(10-120 mM) has been proposed to inhibit the isomerization of asparticacid to isoaspartic acid (WO 2004/039337).

Metal ions were found to confer stability and/or increased activity in aformulation of human deoxyribonuclease (rhDNase, Pulmozyme®). Ca²⁺ on(up to 100 mM) increased the stability of the enzyme through a specificbinding site (Chen et al. (1999), J Pharm Sci. 88(4): 477-82). In fact,removal of calcium ions from the solution with EGTA caused an increasein deamidation and aggregation. However, this effect was observed onlywith Ca⁺² ions; other divalent cations—Mg⁺², Mn⁺² and Zn⁺²—were observedto destabilize rhDNase.

Similar effects were observed in formulation of Factor VIII. Ca⁺² andSr⁺² ions stabilized the protein while others like Mg⁺², Mn⁺² and Zn⁺²,Cu⁺² and Fe⁺² destabilized it (Fatouros, et al. (1997), Int. J. Pharm.,155, 121-131). In a separate study with Factor VIII, a significantincrease in aggregation rate was observed in the presence of ions(Derrick et al. (2004), J. Pharm. Sci., 93(10): 2549-57). The authorsnote that other excipients like buffer salts are often contaminated withAl⁺³ ions and illustrate the need to use excipients of appropriatequality in formulated products. Vaccines containing live or killedattenuated picornaviruses, such as Hepatitis A and polio, areconformationally stabilized by magnesium. Metal ions such as calcium,magnesium and zinc improve the stability of oxytocin in an aqueoussolution. Insulin can bind zinc, leading to the formation of dimers andhexamers in a crystalline form, which allows for the preparation ofdifferent formulations with different in vivo release profiles. Thechemical and thermal stability of the hexamer insulin formulation variesin the presence of different levels of zinc and phenol.

Specific Ligands

One approach to improve the conformational stability of proteintherapeutic drugs is to take advantage of the protein's inherent ligandbinding sites. For example, Pulmozyme® not only requires bivalent metalions for its enzymatic activity, it has improved conformationalstability in the presence of calcium ions. Both acidic and basicfibroblast growth factors (aFGF and bFGF) have been evaluated clinicallyfor their ability to promote wound healing, and both proteins naturallybind to the highly negatively charged proteoglycans on cell surfaces. Avariety of other highly negatively charged compounds also bind anddramatically stabilize aFGF by interaction with the protein's polyanionbinding site.

Surfactants

Protein molecules have a high propensity to interact with surfaces,making them susceptible to adsorption and denaturation at air-liquid,vial-liquid, and liquid-liquid (silicone oil) interfaces. Thisdegradation pathway is inversely dependent on protein concentration andresults in soluble or insoluble protein aggregates or the loss ofprotein from solution through adsorption to surfaces. In addition tocontainer surface adsorption, surface-induced degradation is exacerbatedwith physical agitation, as would be experienced during shipping andhandling.

Surfactants are commonly used in protein formulations to preventsurface-induced degradation. Surfactants are amphipathic molecules withthe capability of out-competing proteins for interfacial positions.Hydrophobic portions of the surfactant molecules occupy interfacialpositions (e.g., air/liquid), while hydrophilic portions of themolecules remain oriented towards the bulk solvent. At sufficientconcentrations (typically around the detergent's critical micellarconcentration), a surface layer of surfactant molecules serve to preventprotein molecules from adsorbing at the interface. Thereby,surface-induced degradation is minimized.

The most commonly used surfactants are the non-ionic fatty acid estersof sorbitan polyethoxylates—i.e., polysorbate 20 and polysorbate 80(e.g., in the drug products Avonex®, Neupogen®, Neulasta®). The twodiffer only in the length of the aliphatic chain that impartshydrophobic character to the molecules, C-12 and C-18, respectively.Polysorbate 80 is more surface-active and has a lower critical micellarconcentration than polysorbate 20. Both polysorbate 20 and polysorbate80 have been shown to protect against agitation-induced aggregation.Polysorbate 20 and 80 also protect against stress induced by freezing,lyophilization and reconstitution. Both polysorbate 20 and 80 maycontain peroxides which can oxidize proteins and they themselves maydegrade by either oxidation or hydrolysis with varying effects onprotein stability. It can also be difficult to control the level ofpolysorbate 20 or 80 in formulations due to their complex behaviorduring membrane filtration (especially at concentrations in whichpolysorbates form micelles in solution). The surfactant poloxamer 188has also been used in several marketed liquid products, such Gonal-F®,Norditropin®, and Ovidrel®, it is generally believed that non-ionicsurfactants stabilize proteins primarily by outcompeting proteinmolecules for hydrophobic surfaces (e.g., airwater interfaces), therebypreventing proteins from unfolding at these hydrophobic interfaces.Non-ionic surfactants can also block protein molecules from adsorbing toother hydrophobic surfaces present during processing. In addition,non-ionic surfactants may directly interact with hydrophobic regions inprotein molecules. Monoclonal antibodies can affect the critical micelleconcentration of polysorbate 20 compared to buffer alone.

Detergents can also affect the thermodynamic conformational stability ofproteins. Here again, the effects of a given excipient will beprotein-specific. For example, polysorbates have been shown to reducethe stability of some proteins and increase the stability of others.Detergent destabilization of proteins can be rationalized in terms ofthe hydrophobic tails of the detergent molecules that can engage inspecific binding with partially or wholly unfolded protein states. Thesetypes of interactions could cause a shift in the conformationalequilibrium towards the more expanded protein states (i.e., increasingthe exposure of hydrophobic portions of the protein molecule incomplement to binding polysorbate). Alternatively, if the protein nativestate exhibits some hydrophobic surfaces, detergent binding to thenative state may stabilize that conformation.

Another aspect of polysorbates is that they are inherently susceptibleto oxidative degradation. Often, as raw materials, they containsufficient quantities of peroxides to cause oxidation of protein residueside-chains, especially methionine. The potential for oxidative damagearising from the addition of stabilizer emphasizes the point that thelowest effective concentrations of excipients should be used informulations. For surfactants, the effective concentration for a givenprotein will depend on the mechanism of stabilization. It has beenpostulated that if the mechanism of surfactant stabilization is relatedto preventing surface denaturation, then the effective concentrationwill be around the detergent's critical micellar concentration.Conversely, if the mechanism of stabilization is associated withspecific protein-detergent interactions, the effective surfactantconcentration will be related to the protein concentration and thestoichiometry of the interaction (Randolph et al. (2002). PharmBiotechnol., 13:159-75).

Surfactants may also be added in appropriate amounts to preventsurface-related aggregation during freezing and drying (Chang (1996), J.Pharm. Sci. 85:1325). Exemplary surfactants include anionic, cationic,nonionic, zwitterionic, and amphoteric surfactants, includingsurfactants derived from naturally occurring amino acids. Anionicsurfactants include, but are not limited to, sodium lauryl sulfate,dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate,chenodeoxycholic acid, N-lauroylsarcosine sodium salt, lithium dodecylsulfate, 1-octanesulfonic acid sodium salt, sodium cholate hydrate,sodium deoxycholate, and glycodeoxycholic acid sodium salt. Cationicsurfactants include, but are not limited to, benzalkonium chloride orbenzethonium chloride, cetylpyridinium chloride monohydrate, andhexadecyltrimethylammonium bromide. Zwitterionic surfactants include,but are not limited to, CHAPS, CHAPSO, SB3-10, and SB3-12. Non-ionicsurfactants include, but are not limited to, digitonin, Triton X-100,Triton X-114, TWEEN-20, and TWEEN-80. In another embodiment, surfactantsinclude lauromaerogol 400, polyoxyl 40 stearate, polyoxyedylenehydrogenated castor oil 10, 40, 50 and 60, glycerol monostearate,polysorbate 40, 60, 65 and 80, soy lecithin and other phospholipids suchas DOPC, DMPG, DMPC, and DOPG; sucrose fatty acid ester, methylcellulose and carboxymethyl cellulose.

Salts

Salts are often added to increase the ionic strength of the formulation,which can be important for protein solubility, physical stability, andisotonicity. Salts can affect the physical stability of proteins in avariety of ways. Ions can stabilize the native state of proteins bybinding to charged residues on the protein's surface. Alternatively,they can stabilize the denatured state by binding to the peptide groupsalong the protein backbone (—CONH—). Salts can also stabilize theprotein native conformation by shielding repulsive electrostaticinteractions between residues within a protein molecule. Electrolytes inprotein formulations can also shield attractive electrostaticinteractions between protein molecules that can lead to proteinaggregation and insolubility.

The effect of salt on the stability and solubility of proteins variessignificantly with the characteristics of the ionic species. TheHofmeister series originated in the 1880's as a way to rank orderelectrolytes based on their ability to precipitate proteins (Cacace etal. (1997), Quarterly Reviews of Biophysics, 30(3): 241-277). In thisreport, the Hofmeister series is used to illustrate a scale of proteinstabilization effects by ionic and non-ionic co-solutes. In Table C,co-solutes are ordered with respect to their general elects on solutionstate proteins, from stabilizing (kosmotropic) to destabilizing(chaotropic). In general, the differences in effects across the anionsare far greater than that observed for the cations, and, for both types,the effects are most apparent at higher concentrations than areacceptable in parenteral formulations. High concentrations ofkostnotropes (c,g, >1 molar ammonium sulfate) are commonly used toprecipitate proteins front solution by a process called ‘salting-out’where the kosmotrope preferentially excluded front the protein surfacereducing the solubility of the protein in it's native (folded)conformation. Removal or dilution of the salt will return the protein tosolution.

The term ‘salting-in’ refers to the use of destabilizing ions (e.g.,like, guanidine and chloride) that increase the solubility of proteinsby solvating the peptide bonds of the protein backbone. Increasingconcentrations of the chaotrope will favor the denatured (unfolded)state conformation of the protein as the solubility of the peptide chainincreases. The relative effectiveness of ions to ‘salt-in’ and‘salt-out’ defines their position in the Hofmeister series.

TABLE 4 The Hofmeister series of salts Cosolute Anion Cation OtherStabilization Scales F⁻ PO₄ ⁻ SO₄ ⁻ CHCOO⁻ Cl⁻ Br⁻ I⁻       (CH₃)₄N⁺(CH₃)₂NH⁺ NH₄ ⁺ K⁺ Na⁺ Ca⁺ Li⁺ Mg²⁺ Ca²⁺ Ba²⁺ Glycerol/SorbitolSucrose/Trehalose TMAO         Guanidine Arginine Urea

In order to maintain isotonicity in parenteral formulation, saltconcentrations are generally limited to less than 150 mM for monovalention combinations. In this concentration range, the mechanism of saltstabilization is probably due to screening of electrostatic repulsiveintramolecular forces or attractive intermolecular forces (Debye-Huckelscreening). Interestingly, chaotropic salts have been shown to be moreeffective at stabilizing the protein structure than similarconcentrations of kosmotropes by this mechanism. The chaotropic anionsare believed to bind more strongly than the kosmotropic ions. Withrespect to covalent protein degradation, differential effects of ionicstrength on this mechanism are expected through Debye-Huckel theory.Accordingly, published reports of protein stabilization by sodiumchloride are accompanied by those where sodium chloride acceleratedcovalent degradation. The mechanisms by which salts affect proteinstability are protein specific and may vary significantly as a functionof solution pH. An example where an excipient can be useful in enablingthe delivery of a protein drug is that of some high concentrationantibody formulations. Over the last several years, salts have beenshown to be effective in reducing the viscosity of such formulations(Liu et al. (2005, 2006), J. Pharm Sci., 94(9): 1928-40, erratum in JPharm Sci. 950): 234-5.

Preservatives

Preservatives are necessary when developing multi-use parenteralformulations that involve more than one extraction from the samecontainer. Their primary function is to inhibit microbial growth andensure product sterility throughout the shelf life or term of use of thedrug product. Commonly used preservatives include benzyl alcohol, phenoland m-cresol. Although preservatives have a long history of use, thedevelopment of protein formulations that includes preservatives can bechallenging. Preservatives almost always have a destabilizing effect(aggregation) on proteins, and this has become a major factor inlimiting their use in multi-dose protein formulations (Roy et al.(2005), J. Pharm. 94(2): 382-96). Benzyl alcohol has also been shown toaffect protein structure and stability in a concentration-, temperature-and time-dependent manner. Due to these destabilizing effects, manylyophilized protein formulations are reconstituted with diluentcontaining benzyl alcohol to minimize the contact time with the proteinprior to administration.

Most protein drugs have been formulated for single-use only. However,when multi-dose formulations are possible, they have the added advantageof enabling patient convenience, and increased marketability. A goodexample is that of human growth hormone (hGH) where the development ofpreserved formulations has led to commercialization of more convenient,multi-use injection pen presentations. At least four such pen devicescontaining preserved formulations of hGH are currently available.Norditropin® (liquid), Nutropin AQ® (liquid) & Genotropin(lyophilized—dual chamber cartridge) contain phenol while Somatrope® isformulated with m-cresol.

Several aspects need to be considered during the formulation developmentof preserved dosage forms. The effective preservative concentration inthe drug product must be optimized. This requires testing a givenpreservative in the dosage form with concentration ranges that conferanti-microbial effectiveness without compromising protein stability. Forexample, three preservatives were successfully screened in thedevelopment of a liquid formulation for interleukin-1 receptor (Type 1),using differential scanning calorimetry (DSC). The preservatives wererank-ordered based on their impact on stability at concentrationscommonly used in marketed products (Remmele et al. (1998), Pharm. Res.,15(2): 200-8).

As might be expected, development of liquid formulations containingpreservatives are more challenging than lyophilized formulations.Freeze-dried products can be lyophilized without the preservative andreconstituted with a preservative containing diluent at the time of use.This shortens the time during which a preservative is in contact withthe protein, thus significantly minimizing the associated stabilityrisks. With liquid formulations, preservative effectiveness andstability have to be maintained over the entire product shelf-life(usually about 18-24 months). An important point to note is thatpreservative effectiveness has to he demonstrated in the finalformulation containing the active drug and all excipient components.

Some preservatives can cause injection site reactions, which is anotherfactor that needs consideration when choosing a preservative, inclinical trials that focused on the evaluation of preservatives andbuffers in Norditropin®, pain perception was observed to be lower informulations containing phenol and benzyl alcohol as compared to aformulation containing m-cresol (Kappelgaard (2004), Horm. Res. 62 Suppl3:98-103). Interestingly, among the commonly used preservative, benzylalcohol possesses anesthetic properties (Minogue and Sun (2005), Anesth.Analg. 100(3): 683-6).

WORKING EXAMPLES

All publications, patents, and patent applications discussed and citedherein are hereby incorporated by reference m their entireties. It isunderstood that the disclosed invention is not limited to the particularmethodology, protocols and materials described as these can vary. It isalso understood that the terminology used herein is for the purposes ofdescribing particular embodiments only and is not intended to limit thescope of the appended claims.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the claims that follow.

Example 1

This experiment demonstrates that by adjusting fill weight targets,protein concentration can be precisely targeted for the respectivereconstituted drug product.

Materials

-   -   Buffer containing: Mannitol, Sucrose, L-Histidine, Polysorbate        20 at pH 5    -   Container Vial, 3 cc, Blowback, Type I Glass, Non-treated, 13 mm        Finish with Stopper, 13 mm, 4432/50 V-50,    -   romiplostim Filtered Purified Bulk

Method

-   -   1. Dilute drug to target product formulation (0.5 mg/mL)        utilizing required amount of the dilution buffer.    -   2. Filter formulated solution using a 0.22 μm Polyvinylidene        difluoride (PVDF) filter.    -   3. Ensure vials and stopper: 3 cc vials have been washed and        depyrogenated.    -   4. Fill sufficient quantity of vials to respective fill weight        targets: 0.307, 0.322, 0.342, 0.357, 0.373 g.    -   5. Partially stopper vials and place in lyophilizer.    -   6. Run required lyophilization cycle with adequate freezing,        vacuum, with primary and secondary drying, followed by        stoppering and unloading of the lyophilized product in sealed        vials.    -   7. Reconstitute product with set reconstitution volume of 0.32        mL of water for injection.    -   8. Measure resulting protein concentration in vials utilizing        ultraviolet (UV) absorption, Absorbance is defined as the amount        of light of specific wavelength that is absorbed as it passes        through an analyte. The Absorbance Unit is a function of the        intrinsic absorbance of the molecule, its concentration and the        path length of the analyte. Aromatic amino acids phenylalanine,        tyrosine, and tryptophan in protein molecules absorb light in        the UV range of 260-290 nm. UV absorption in this range is used        routinely to measure protein presence in a solution.    -   9. Measure the osmolality of the product utilizing freezing        point depression.    -   10. Perform analysis to determine effect of adjusted fill weight        targets for both protein and osmoality.

TABLE 5 Summary of fill weights and protein concentration ExperimentalParameters conditions A B C D E Input Formulated bulk 0.528 0.528 0.5280.528 0.528 concentration (mg/mL) Average fill weight (g) 0.307 0.3220.342 0.357 0.373 Output Average protein 0.459 0.484 0.518 0.546 0.565concentration post- reconstitution (mg/mL)

A strong linear relationship between reconstituted protein concentrationand fill volume was observed as evidenced by the R² of 0.9964 as shownin FIG. 6. This linear relationship can be explained theoretically basedon protein mass balance, as shown below:

$\begin{matrix}{{C_{reconstitution}*V_{reconstitution}} = {\left( {C_{Formulated} - C_{loss}} \right)*V_{fill}}} & \left\lbrack {{Equation}\mspace{14mu} 1} \right\rbrack \\{C_{reconstitution} = {\frac{C_{formulated} - C_{loss}}{V_{reconstitution}}*V_{fill}}} & \left\lbrack {{Equation}\mspace{14mu} 2} \right\rbrack\end{matrix}$

Where C_(reconstitution) is the protein concentrationpost-reconstitution, V_(reconstitution) is the product volumepost-reconstitution, C_(formatted m)is the formulated bulk proteinconcentration, C_(loss) is the protein loss due to binding to filter andcontainers, and V_(fill) is the fill volume in each vial. Sinceformulated bulk concentration, protein binding loss and reconstitutionvolume are constant in a given run, protein concentrationpost-reconstitution is proportional to fill volume as indicated inEquation 2.

In the pilot scale experiment, variability of final reconstituted drugproduct (DP) protein concentration was determined using 10 replicates ateach filling condition, as shown in Table 6. Therefore, the observedvariability represents both process and analytical variability,including the variability associated with reconstitution of the product.

TABLE 6 Protein concentration (reconstituted) variability under eachfilling condition A B C D E (−10%) (−5%) (Target) (+5%) (+10%) Number of10 10 10 10 10 replicates Average (mg/ 0.459 0.484 0.518 0.546 0.565 ml)Standard 0.0084 0.0133 0.0082 0.0052 0.0069 deviation (SD, mg/mL)Relative SD (%) 1.83 2.76 1.58 0.94 1.21

Fill Weight Impact on Osmolality (Reconstituted DP)

The final drug product osmolality after reconstitution was tested forosmolality, for vials filled at five fill weight targets. The osmolalityresults before filling and post reconstitution are summarized in Table7. The osmolality doesn't change post filling and reconstitution basedon the results at the target fill weight of 0.341 grams, indicating thatthe excipients were not lost in appreciable amounts. Osmolalityincreases slightly at higher fill volumes, and decreases slightly atlower fill volumes when reconstitution volume is held constant.

TABLE 7 Summary of Fill weights and Osmolality Parameters A B C D EInput Formulated bulk 312.4 312.4 312.4 312.4 312.4 osmolality(mOsm/kg)* Average fill weight (g) 0.307 0.322 0.342 0.357 0.373 Averagefill volume (mL) 0.301 0.316 0.336 0.350 0.366 Normalized volume (%)89.9% 94.3% 100.2% 104.6% 109.3% Output Effective volume post 0.3350.335 0.335 0.335 0.335 reconstitution (mL) Average osmolality post 280294 313 327 342 reconstitution (mOsm/kg) Normalized osmolality 89.6%94.0% 100.0% 104.6% 109.3% (%)

A linear relationship was found if the fill weight/volume (normalizedbased on target of 0.341 g) is plotted versus the product osmolality(normalized based on target osmolality of 313 mOsm/kg at target fillweight), as shown in FIG. 7. This linear relationship can be explainedby the definition of osmolality and its relationship with buffercomponent concentration

Osmolality is a measure of solute concentration, defined as the numberof osmoles (Osm) of solute per kilogram of solvent (osmo/kg or Osm/kg).Distinct from molarity (mole/L), osmolality measures moles of soluteparticles (such as dissociated ion) rather than moles of solute.Osmolality of a solution can be calculated from the followingexpression:

Osmolality (osm/kg)=density*Σφ_(i) n _(i) C _(i)   [Equation 3]

Where φ is the osmotic coefficient, n is the number of particles (e.g.,ions) into which a molecule dissociates, and C is the molarconcentration (mole/L) of the solute.

In the case when fill weight (or volume) is 10% higher than targetvolume, the concentration of each excipient species in the formulationis increased by 10% upon reconstitution to constant volume. Since φ_(i)and n_(i) are constant in a known formulation, 10% increase inconcentration of each species results in 10% increase in osmolality asshown in Equation 3. Similarly, 10% decrease of fill volume results in10% decrease in excipient concentration, and consequently 10% decreasein osmolality.

Example 2

The protein blinatumomab has 55 μg/mL, formulated to 200 mML-lysine-HCl, 25 mM citric acid, 15% (w/v) trehalose dihydrate, 0.1%(w/v) polysorbate 80, pH 7.0. The formulated protein allowable range ismeasured at the filtered bulk stage. Bulk concentrations ranging from48.0 μg/mL to 65.0 μg/mL are used. Target fill weights are calculatedbased upon the measured protein concentration to target a reconstituteddrug product of 12.5 mcg/mL when reconstituted with 3 mL of water.

C _(reconstitution) *V _(reconstitution)=(C _(Formulated) −C _(loss))*V_(fill)

Where

C _(reconstitution) *V _(reconstitution)=Target proteincontent_(reconstitution)

Then the Target protein content can then be multiplied by the productdensity and divided by the measured drug concentration (adjusted forloss due to binding if needed) to determine the Target fill weight.

The target fill weight is calculated according to the following formula,with a corresponding fill weight range of 0.634 to 0.858 gm.

${{Fill}\mspace{14mu} {weight}\mspace{14mu} (g)} = \frac{{target}\mspace{14mu} {dose}\mspace{14mu} \left( {38.5\mspace{14mu} µ\frac{g}{vial}} \right) \times {{density}\left( {1.07\mspace{14mu} \frac{g}{mL}} \right)}}{{DS}\mspace{14mu} {concentration}\mspace{14mu} \left( {x\mspace{14mu} µ\frac{g}{mL}} \right)}$

wherein DS is generally understood by persons of ordinary skill in theart to refer to drug substance. Stated more generally,

${{adjusted}\mspace{14mu} {fill}\mspace{14mu} {weight}} = {\frac{\left( {{target}\mspace{14mu} {fixed}\mspace{14mu} {dose}\mspace{14mu} {of}\mspace{14mu} {the}\mspace{14mu} {therapeutic}\mspace{14mu} {protein}} \right) \times ({density})}{{therapeutic}\mspace{14mu} {protein}\mspace{14mu} {concentration}\mspace{14mu} {in}\mspace{11mu} {bulk}\mspace{14mu} {formulation}}.}$

Example 3

Infliximab drug product has 20±1.5 mg/mL infliximab, formulated with 10mM sodium phosphate, 10% (w/v) sucrose, 0.01% (w/v) polysorbate 80, pH7.2 post reconstitution with 10 mL Water for Injection.

The target fill weight is calculated according to following formula,with a corresponding fill weight range of 4.85 to 5.63 gm.

Calculation  of  target  fill  weight${{Target}\mspace{14mu} {fill}\mspace{14mu} {weight}\mspace{14mu} (g)} = \frac{\left( {{Target}\mspace{14mu} {protein}\mspace{14mu} {content}\mspace{14mu} \left( {100\mspace{14mu} {mg}} \right)} \right) \times \left( {{Density}\mspace{14mu} \left( {1.042\mspace{14mu} \frac{g}{mL}} \right)} \right)}{{Released}\mspace{14mu} {drug}\mspace{14mu} {substance}\mspace{14mu} {protein}\mspace{14mu} {concentration}\mspace{14mu} \left( \frac{mg}{mL} \right)}$

Example 4

Trastuzumab has 21 mg/mL formulated with, 0303 mg/mL L-Histidine, 0.470mg/mL L-Histidine Hydrochloride Monohydrate, 19.1 mg/mL α,α-TrehaloseDihydrate, 0.0840 mg/mL Polysorbate 20, at pH 6.1. The target fillweight is calculated according to formula below, with varying fillweight ranges based upon the product delivery requirements. The fillweight targets range from 3.1 to 21.2 gm (depending upon the respectivepresentation). This product has multiple presentations with a pooleddrug substance concentration of 21 mg/mL.

The target fill weight for each drug product lot for this Example 4 andthe subsequent examples is calculated using the following information:

${{Target}\mspace{14mu} {fill}\mspace{14mu} {{weight}\mspace{14mu}\lbrack g\rbrack}} = \frac{\left( {{Target}\mspace{14mu} {protein}\mspace{14mu} {{content}\mspace{14mu}\lbrack{mg}\rbrack}} \right) \times \left( {{Density}\mspace{14mu}\left\lbrack {g\text{/}{mL}} \right\rbrack} \right)}{{Pooled}\mspace{14mu} {drug}\mspace{14mu} {substance}\mspace{14mu} {{concentration}\mspace{14mu}\left\lbrack {{mg}\text{/}{mL}} \right\rbrack}}$

As an example,

${{the}\mspace{14mu} {target}\mspace{14mu} {fill}\mspace{14mu} {weight}\mspace{14mu} {for}\mspace{14mu} a\mspace{14mu} 150\mspace{14mu} {mg}\mspace{14mu} {{presentation}\mspace{14mu}\lbrack g\rbrack}} = \frac{\left( {156\mspace{14mu} {mg}} \right) \times \left( {1.01\mspace{14mu} g\text{/}{mL}} \right)}{{Pooled}\mspace{14mu} {drug}\mspace{14mu} {substance}\mspace{14mu} {{concentration}\mspace{14mu}\left\lbrack {21\mspace{14mu} {mg}\text{/}{mL}} \right\rbrack}}$

As another example,

${{the}\mspace{14mu} {target}\mspace{14mu} {fill}\mspace{14mu} {weight}\mspace{14mu} {for}\mspace{14mu} a\mspace{14mu} 420\mspace{14mu} {mg}\mspace{14mu} {{presentation}\mspace{14mu}\lbrack g\rbrack}} = \frac{\left( {440\mspace{14mu} {mg}} \right) \times \left( {1.01\mspace{14mu} g\text{/}{mL}} \right)}{{Pooled}\mspace{14mu} {drug}\mspace{14mu} {substance}\mspace{14mu} {{concentration}\mspace{14mu}\left\lbrack {21\mspace{14mu} {mg}\text{/}{mL}} \right\rbrack}}$

As a further example, the

${{the}\mspace{14mu} {target}\mspace{14mu} {fill}\mspace{14mu} {weight}\mspace{14mu} {for}\mspace{14mu} a\mspace{14mu} 60\mspace{14mu} {mg}\mspace{14mu} {{presentation}\mspace{14mu}\lbrack g\rbrack}} = \frac{\left( {65\mspace{14mu} {mg}} \right) \times \left( {1.01\mspace{14mu} g\text{/}{mL}} \right)}{{Pooled}\mspace{14mu} {drug}\mspace{14mu} {substance}\mspace{14mu} {{concentration}\mspace{14mu}\left\lbrack {21\mspace{14mu} {mg}\text{/}{mL}} \right\rbrack}}$

Example 5

AMG 701 is a single chain, variable domain Bi-specific T-cell Engager(BiTE®) anti-BCMA/anti-CD3 antibody construct (see NCI Drug Dictionaryand other references). AMG 701 with protein concentration at 1 mg/mL wasformulated with 10 mM L-glutamic acid, 9.0% (w/v) sucrose, 0.010% (w/v)polysorbate 80, at pH 4.2. The target fill weight is calculatedaccording to formula below, with varying fill weight ranges based uponthe product delivery requirements. AMG 701 has three presentations, withthe fill weight targets range for the first presentation from 0.47 to0.57 gm, the second presentation from 1.60 to 1.96 gm, and the thirdpresentation from 3.28 to 4.01 gm.

For the first presentation:

${{Target}\mspace{14mu} {fill}\mspace{14mu} {weight}\mspace{14mu} (g)} = \frac{\left( {{Target}\mspace{14mu} {protein}\mspace{14mu} {content}\mspace{14mu} \left( {0.50\mspace{14mu} {mg}} \right)} \right) \times \left( {{Density}\mspace{14mu} \left( {1.032\mspace{14mu} \frac{g}{mL}} \right)} \right)}{{Released}\mspace{14mu} {drug}\mspace{14mu} {substance}{\mspace{11mu} \;}{protein}\mspace{14mu} {{concentration}{\mspace{11mu} \;}\left( {1.0\mspace{14mu} \frac{mg}{mL}} \right)}}$

For the second presentation:

${{Target}\mspace{14mu} {fill}\mspace{14mu} {weight}\mspace{14mu} (g)} = \frac{\left( {{Target}\mspace{14mu} {protein}\mspace{14mu} {content}\mspace{14mu} \left( {1.71\mspace{14mu} {mg}} \right)} \right) \times \left( {{Density}\mspace{14mu} \left( {1.032\mspace{14mu} \frac{g}{mL}} \right)} \right)}{{Released}\mspace{14mu} {drug}\mspace{14mu} {substance}{\mspace{11mu} \;}{protein}\mspace{14mu} {{concentration}{\mspace{11mu} \;}\left( {1.0\mspace{14mu} \frac{mg}{mL}} \right)}}$

For the third presentation:

${{Target}\mspace{14mu} {fill}\mspace{14mu} {weight}\mspace{14mu} (g)} = \frac{\left( {{Target}\mspace{14mu} {protein}\mspace{14mu} {content}\mspace{14mu} \left( {3.5\mspace{14mu} {mg}} \right)} \right) \times \left( {{Density}\mspace{14mu} \left( {1.032\mspace{14mu} \frac{g}{mL}} \right)} \right)}{{Released}\mspace{14mu} {drug}\mspace{14mu} {substance}{\mspace{11mu} \;}{protein}\mspace{14mu} {{concentration}{\mspace{11mu} \;}\left( {1.0\mspace{14mu} \frac{mg}{mL}} \right)}}$

Example 6

AMG 330 is an anti-CD33/anti-CD3 single chain, variable domainBi-specific T-cell Engager (BiTE®) (see NCI Drug Dictionary and otherreferences). AMG 300 with protein concentration at 0.5 mg/mL wasformulated with 10 mM potassium phosphate, 8.0% (w/v) sucrose, 1.0%(w/v) sulfobutylether betacyclodextrin (SBE-CD), 0.010% (w/v)polysorbate 80 at pH 6.1. The target fill weight is calculated accordingto formula below, with varying fill weight ranges based upon the productdelivery requirements. The fill weight targets range from 1.2 to gm. Thetarget fill weight is calculated as follows:

${{Target}\mspace{14mu} {fill}\mspace{14mu} {weight}\mspace{14mu} (g)} = \frac{\left( {{Target}\mspace{14mu} {protein}\mspace{14mu} {content}\mspace{14mu} \left( {0.64\mspace{14mu} {mg}} \right)} \right) \times \left( {{Density}\mspace{14mu} \left( {1.033\mspace{14mu} \frac{g}{mL}} \right)} \right)}{{Drug}\mspace{14mu} {substance}{\mspace{11mu} \;}{protein}\mspace{14mu} {{concentration}{\mspace{11mu} \;}\left( {0.5\mspace{14mu} \frac{mg}{mL}} \right)}}$

In general, the methodology can be used in the case where a targetamount of product is required in the container to ensure that theproduct post-reconstitution is at the required concentration. Thisresult is achieved by determining the amount of volume of thereconstituted product; for example, 1 mL volume with a desiredconcentration of 1 mg/mL therapeutic protein, leading to a total amountof required protein content of 1 mg.

Target protein content [mg]=(Protein concentration [mg/mL]×Reconstitutedvolume [mL])

The target protein content is then used to calculate the target fillweight based upon the formula below. The in-process or measured drugconcentration is typically measured as part of the formulation processto compensate for any process variability. In some cases, an adjustmentto the target protein content is made to compensate for loss of productdue to binding. Confidence in the measured drug concentration isrequired to ensure precise targeting of the fill weights.

${{Target}\mspace{14mu} {fill}\mspace{14mu} {{weight}\lbrack g\rbrack}} = \frac{\left( {{Target}\mspace{14mu} {protein}\mspace{14mu} {{content}\lbrack{mg}\rbrack}} \right) \times \left( {{Density}\left\lbrack {g\text{/}{mL}} \right\rbrack} \right)}{{Measured}\mspace{14mu} {drug}\mspace{14mu} {{concentration}\left\lbrack {{mg}\text{/}{mL}} \right\rbrack}}$

This is a specific example as the therapeutic product both for thein-process concentration and the reconstituted product can range frommicrograms (mcg or μg) per milliliter (mL) to milligrams (mg) permilliliter (mL).

Verification of osmolality is required, as discussed above (based uponexperimental findings). Limitations on the allowed measured drugconcentration are established in combination with the fill weighttargets to achieve the required amount of active drug filled into thecontainer to provide assurance that the reconstituted product meets boththe product (active drug or protein) concentration as well as theosmolality specification limits.

All publications, patents, and patent applications discussed and citedherein are hereby incorporated by reference in their entireties. It isunderstood that the disclosed invention is not limited to the particularmethodology, protocols and materials described as these can vary. It isalso understood that the terminology used herein is for the purposes ofdescribing particular embodiments only and is not intended to limit thescope of the appended claims.

Those skilled in the art will recognize or be able to ascertain using nomore than routine experimentation many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the claims that follow.

What is claimed is:
 1. A process for making a lyophilized pharmaceuticalfor of therapeutic protein, which comprises: a. providing a formulationof a bulk amount of the therapeutic protein, b. measuring theconcentration of the therapeutic protein in said bulk formulation, c.adjusting the fill weight of the protein in said bulk formulation toachieve a fixed dose of the protein, and d. lyophilizing the proteinfill weight-adjusted formulation to achieve a final formulation in acontainer, wherein the product concentration post reconstitution with afixed volume is within a predetermined acceptance range.
 2. The processof claim 1, wherein the adjusted fill weight of the protein iscalculated according to the formula${{adjusted}\mspace{14mu} {fill}\mspace{14mu} {weight}} = {\frac{\left( {{target}\mspace{14mu} {fixed}\mspace{14mu} {dose}\mspace{14mu} {of}\mspace{14mu} {the}{\mspace{11mu} \;}{therapeutic}\mspace{14mu} {protein}} \right) \times ({density})}{{therapeutic}\mspace{14mu} {protein}\mspace{14mu} {concentration}\mspace{14mu} {in}\mspace{14mu} {bulk}\mspace{14mu} {formulation}}.}$3. The process of claim 1, wherein the protein concentration in thefinal formulation is less than or equal to about 20 mg/mL.
 4. Theprocess of claim 1, wherein the therapelitic protein is selected fromromiplostim, blinatumomab, infliximab, trastuzumab, AMG 701, and AMG330.
 5. The process of claim 1, wherein the therapeutic protein isromiplostim and the therapeutic protein concentration in the finalformulation is about 0.5 mg/mL.
 6. The process of claim 1, wherein thefinal formulation comprises about 0.5 mg/mL romiplostim in about 10 mMhistidine, about 4% w/v mannitol, about 2% w/v sucrose and about 0.004%polysorbate 20 at about pH 5.0.
 7. The process of claim 1, wherein thetherapeutic protein is blinatumomab and the therapeutic proteinconcentration in the final formulation is about 55 μg/mL.
 8. The processof claim 1, wherein the formulation comprises about 55 μg/mLblinatumomab in about 25 mM citric acid monohydrate, about 15% (w /v)trehalose, about 200 mM L-lyse hydrochloride, and about 0.1% (w/v)polysorbate 80 at about pH 7.0.
 9. The process of claim 1, wherein thetherapeutic protein is infliximab and the therapeutic proteinconcentration in the final formulation is about 20±1.5 mg/mL.
 10. Theprocess of claim 1, wherein the final form elation comprises about20±1.5 mg/mL infliximab, about 10 mM sodium phosphate, about 10% (w/v)sucrose and about 0.01% (w/v) polysorbate 80 at about pH 7.2.
 11. Theprocess of claim 1, wherein the therapeutic protein is trastuzumab andthe therapeutic protein concentration in the final formulation is about21 mg/mL.
 12. The process of claim 1, wherein the final formulationcomprises about 21 mg/mL trastuzumab, about 0.303 mg/mL L-histidine,about 0.470 mg/ml L-histidine hydrochloride monohydrate, about 19.1mg/mL α,α-trehalose dihydrate, and about 0.0840 mg/mL polysorbate 20 atabout pH 6.1.
 13. The process of claim 1, wherein the therapeuticprotein is AMG 701 and the therapeutic protein concentration in thefinal formulation is about 1 mg/mL.
 14. The process of claim 1, whereinthe final formulation comprises about 1 mg/mL AMG 701, about 10 mML-glutamic acid, about 9.0% (w/v) sucrose, and about 0.010% (w/v)polysorbate 80 at about pH 4.2.
 15. The process of claim 1, wherein thetherapeutic protein is AMG 330 and the therapeutic protein concentrationin the final formulation is about 0.5 mg/mL.
 16. The process of claim 1,wherein the final formulation comprises about 0.5 AMG 330, about 10 mMpotassium phosphate, about 8.0% (w/v) sucrose, about 1.0% (w/v)sulfobutylether betacyclodextrin (SBE-CD), and about 0.010% (w/v)polysorbate 80 at about pH 6.1.
 17. The process of claim 1, wherein theprotein concentration in the final formulation is less than or equal toabout 25 mg/mL.
 18. The process of claim 1, wherein the therapeuticprotein is a bispecific single chain antibody construct.
 19. The processof claim 1, wherein the therapeutic protein is selected from AMG 701 andAMG 330.